The present invention relates to carboxyalkyl derivatives of 2-oxo-3-hydroxy-lysergic acid diethylamide (LSD) and to the use of these derivatives to prepare immunogens for stimulating antibody production. The antibodies so produced are useful in an immunoassay for determining LSD and 2-oxo-3-hydroxy-LSD. The invention also relates to a method for antibody screening.
The chemical structure of LSD is 9,10-didehydro-N,N-diethyl-6-methylergoline-8β-carboxamide and can be represented by the formula

LSD is a highly potent hallucinogen, with the typical dosage range being 25 to 150 μg. The drug undergoes rapid and extensive metabolism, and only about 1% of the parent drug is actually excreted in human urine (Poch, G. K. et al., J. Chromatogr. B 724, 23-33, 1999). Possible metabolic transformations may be hydrolysis to lysergic acid, N-demethylation to N-desmethyl-LSD (nor-LSD) and oxidation to 2-oxo-LSD and 2-oxo-3-hydroxy-LSD. Isolysergic diethylamide (iso-LSD) is a byproduct of LSD synthesis and is often detected in the urine from an LSD user because of its presence as a contaminant in LSD sold on the street. The structure of iso-LSD is represented by the formula

LSD is one of the most difficult drugs of abuse to detect in urine because of the very low concentrations of the parent drug excreted in the urine. 2-Oxo-3-hydroxy-LSD is a recently identified metabolite of LSD that has been found to be present in urine from LSD users at concentrations from 4 to 40 times higher than LSD and that can be detected for a longer time than LSD after ingestion of the drug (Reuschel, S. A., et al., J. Anal. Toxicol. 23, 306-312, 1999; Verstraete, A. G., Van de Velde, E. J., Annual Society of Forensic Toxicologists Meeting Scientific Session, Albuquerque, N. Mex., Oct. 5-9, 1998).
In testing for other drugs of abuse, immunoassays, particularly competitive binding immunoassays, have proven to be especially advantageous. In competitive binding immunoassays, an analyte in a biological sample competes with a labeled reagent, or analyte analog, or tracer, for a limited number of receptor binding sites on antibodies specific for the analyte and analyte analog. Enzymes such as β-galactosidase and peroxidase, fluorescent molecules such as fluorescein compounds, and radioactive compounds such as 125I are common labeling substances used as tracers. The concentration of analyte in the sample determines the amount of analyte analog which will bind to the antibody. The amount of analyte analog that will bind is inversely proportional to the concentration of analyte in the sample, because the analyte and the analyte analog each bind to the antibody in proportion to their respective concentrations. The amount of free or bound analyte analog can then be determined by methods appropriate to the particular label being used.
Commercial immunoassay methods for LSD currently available employ monoclonal or polyclonal antibodies specific for LSD and having low cross-reactivity with 2-oxo-3-hydroxy-LSD. For example, the cross-reactivity of 2-oxo-3-hydroxy-LSD in the EMIT (Syva Company), CEDIA (Microgenics Corporation) and KIMS (Roche Diagnostics) immunoassays is 1.7, 1.8 and 11% respectively (Verstraete, A. G., ibid.). The present inventors are unaware of monoclonal antibodies specific for 2-oxo-3-hydroxy-LSD having been reported prior to their invention as described herein.
Haptens are partial or incomplete antigens. They are protein-free substances, mostly low molecular weight substances, which are not capable of stimulating antibody formation, but which do react with antibodies. The latter are formed by coupling the hapten to a high molecular weight carrier and injecting this coupled product into humans or animals. Examples of haptens include therapeutic drugs such as digoxin and theophylline, drugs of abuse such as morphine and LSD, antibiotics such as gentamycin and vancomycin, hormones such as estrogen and progesterone, vitamins such as vitamin B12 and folic acid, thyroxin, histamine, serotonin, adrenaline and others.
An activated hapten refers to a hapten derivative that has been provided with an available site for reaction such as by the attachment of a linking group for synthesizing a derivative conjugate.
A carrier, as the term is used herein, is an immunogenic substance, commonly a protein, that can join with a hapten, thereby enabling the hapten to stimulate an immune response. Carrier substances include proteins, glycoproteins, complex polysaccharides and nucleic acids that are recognized as foreign and thereby elicit an immunologic response from the host.
The terms immunogen and immunogenic as used herein refer to substances capable of producing or generating an immune response in an organism.
The term derivative refers to a chemical compound or molecule made from a parent compound or molecule by one or more chemical reactions.
Linking groups are used to activate, i.e., provide an available site on a drug derivative for synthesizing a hapten. The use of a linking group may or may not be advantageous or needed depending on the specific hapten and carrier pairs. The term linker refers to a chemical moiety that connects a hapten to a carrier, immunogen, label, tracer or another linker. Linkers may be straight or branched, saturated or unsaturated carbon chains. They may also include one or more heteroatoms within the chain or at termini of the chains. By heteroatoms is meant atoms other than carbon which are chosen from the group consisting of oxygen, nitrogen and sulfur.
As used herein, a detector molecule, label or tracer is an identifying tag which, when attached to a carrier substance or molecule, can be used to detect an analyte. A label may be attached to its carrier substance directly or indirectly by means of a linking or bridging moiety. Examples of labels include enzymes such as β-galactosidase and peroxidase, fluorescent compounds such as rhodamine and fluorescein isothiocyanate (FITC), luminescent compounds such as dioxetanes and luciferin, and radioactive isotopes such as 125I.
A peptide is any compound formed by the linkage of two or more amino acids by amide (peptide) bonds, usually a polymer of α-amino acids in which the α-amino group of each amino acid residue (except the NH2-terminal) is linked to the α-carboxyl group of the next residue in a linear chain. The terms peptide, polypeptide and poly(amino acid) are used synonymously herein to refer to this class of compounds without restriction as to size. The largest members of this class are referred to as proteins.
As used herein, oxidized LSD means 2-oxo-3-hydroxy-LSD and 2-oxo-LSD.